Molecular characterisation of Bacillus thuringiensis strain MEB4 highly toxic to the Mediterranean flour moth Ephestia kuehniella Zeller (Lepidoptera: Pyralidae)

Abstract

Background: Cry2 proteins play an essential role in current Bacillus thuringiensis (Bt) applications and in the prevention of insect resistance to Cry1A toxins. This paper reports on the screening and characterisation of novel Bt strains harbouring effective cry2A-type genes and higher insecticidal activity to Ephestia kuehniella.

Results: A total of 29 native Bt strains were screened to search for the potent strain against E. kuehniella. The plasmid pattern of the selected strains showed interesting variability. PCR-RFLP analysis of two amplified regions showed high sequence identity within the selected cry2A-type genes. SDS-PAGE and western blot analysis revealed the presence of Cry2Aa toxin only in the MEB4 and BLB240 strains. The activation of Cry2Aa protoxins by larval midgut juice, trypsin or chymotrypsin enzymes revealed significant differences in terms of proteolysis profiles. Interestingly, a 49 kDa band was detected in the proteolysis pattern of BLB240, suggesting the presence of a chymotrypsin cleavage site that might have affected its insecticidal activity. Further, bioassays demonstrated that MEB4 (103.08 ± 36 µg g(-1)) was more active than BLB240 (153.77 ± 45.65 µg g(-1)) against E. kuehniella.

Conclusion: Based on its potent insecticidal activity, the MEB4 strain could be considered to be an effective alternative agent for the control of E. kuehniella.

Keywords: Bacillus thuringiensis; PCR-RFLP; cry2Aa gene; plasmid pattern; proteolysis.